Our broad goal is to understand anticoagulant mechanisms that regulate blood clotting and prevent unwanted blood clots (thrombosis). These cause pulmonary embolism, stroke, and venous thrombosis, leading to 500,000 hospitalizations and 100,000 deaths a year in the USA. We will focus on mechanisms for the direct antithrombotic activity of protein S (PS-direct), including new mechanisms that are codependent with another anticoagulant protein, tissue factor pathway inhibitor (TFPI). Insights may lead to new treatments, or to measures that prevent thrombosis. Rationale: Protein S was antithrombotic in vivo, independent of APC;PS-direct in plasma was verified;active protein S contained Zn2+ that was crucial for PS-direct, and that was lost during some purification methods;PS-direct was lost/regained with Zn loss/regain;protein S bound to TFPI;interplay with TFPI was shown during tissue factor-based clotting;plasma protein S monomers and multimers had similar ability to inhibit FXa/FVa. Thus, PS-direct is real and health-relevant;protein S deficiency leads to thrombotic risk. Specific Aims: 1. Define coagulation reactions that are sensitive to PS-direct and clarify which are codependent or independent of TFPI. Expand novel plasma and purified component activity assays to find all modes of PS-direct and which ones are TFPI-codependent. Find the requirements for TFPI-codependent PS-direct, e.g., occupation of a Zn site(s) on protein S, Ca, phospholipids, intact thrombin-sensitive loop, or any role of protein S multimers. 2. Determine if protein S binds directly to TFPI, or to an extrinsic FXase component, and find the sites of molecular interaction. Measure binding of protein S to TFPI by physical methods: Test the alternate hypothesis that protein S interacts directly with FXa, tissue factor, or FVIIa and causes a conformation change conducive to their binding to TFPI, or conducive to conversion of inactive Zn-deficient protein S to a metastable active form. Locate the specific protein S binding site involved by use of constructs, peptides, antibodies and limited site-specific mutagenesis. Find the complementary region on TFPI and FXa. 3. Locate a new Zn binding site(s) on protein S that is crucial for PS-direct and find if this site is important for protein S interaction with TFPI, FVa, or FXa. Locate the Zn region through use of existing constructs. Narrow the site using a 3-D model, known Zn-coordinating residues and a limited number of rational site mutants. Measure Zn content and correlate it with PS-direct in modes that are codependent or independent of TFPI. Examine conformation changes. 4. Identify molecular sites crucial for TFPI-independent PS-direct. Define regions/residues implicated in inhibition of FVa and FXa, using existing protein S constructs, mutants, peptides, antibodies. Pinpoint a neutralizing epitope and find if it is involved in FVa or FXa binding. Use a 3-D model to design a limited number of new mutants and to build a cohesive picture of findings. Be alert for potential therapeutic agents that enhance or mimic PS-direct or TFPI. PUBLIC HEALTH RELEVANCE: Our broad goal is to understand anticoagulant mechanisms that regulate blood clotting and prevent unwanted blood clots (thrombosis). These clots cause pulmonary embolism, stroke, and venous thrombosis, leading to 500,000 hospitalizations and 100,000 deaths a year in the USA. We will focus on mechanisms for the direct antithrombotic activity of protein S (PS-direct), including new mechanisms that depend on another anticoagulant protein, tissue factor pathway inhibitor (TFPI). Insights may lead to new treatments, or measures that prevent thrombosis.